【作者】 刘莹莹；

【导师】 祝茜；

【作者基本信息】 山东大学 ， 海洋生物学， 2012， 博士

【摘要】 松江鲈是一种典型的降海洄游性鱼类。历史上,松江鲈分布范围较广。但是,随着环境污染的加剧,松江鲈赖以生存和繁衍的生态环境遭到严重的破坏,现已被列入国家二级保护动物。人工养殖成为恢复其自然种群的重要途径和手段,然而,松江鲈鱼独特的生物学和生理学特性,决定了它与其他鱼类不同的病理、病症特点。面对养殖过程中日益严重的病害问题,深入开展松江鲈免疫机制研究,并在此基础上寻找松江鲈疾病防治的有效方法已成为当务之急。研究表明,生物体在受到病原和重金属等刺激时,因氧化应激体内产生大量的活性氧(Reactive oxygen species, ROS)。ROS在杀灭入侵的病原微生物的同时,由于其反应的非特异性,也会对宿主的细胞、组织和器官造成严重伤害,进而导致机体生理机能的损伤和免疫系统的破坏。所以,消除生物体内因过度免疫反应产生的过量ROS将能够增强其抗病能力,有效地保护免疫器官和组织,抑制免疫细胞的凋亡,提高免疫力。硫氧还蛋白(Thioredoxin, Trx)和过氧化物还原酶(Peroxiredoxin, Prx)分别属于生物体内非酶抗和酶抗氧化剂,在清除ROS过程中发挥重要作用。本论文采用RACE (Rapid amplification of cDNA ends)技术从松江鲈体内克隆到了硫氧还蛋白1(TfTrx1)、硫氧还蛋白相关蛋白14(Thioredoxin related protein14, TfTRP14)和过氧化物还原酶1(TfPrx1)三种与免疫系统相关的抗氧化基因,分析了它们的分子结构特征,采用实时荧光定量PCR (Quantitative real time PCR, qRT-PCR)检测了三个基因的组织分布及LPS和重金属刺激后的表达模式变化；同时,将三个基因连接到pET-30a(+)载体,实现了在大肠杆菌中的重组表达。获得纯化蛋白后,制备多克隆抗体,利用Western blot检测了LPS刺激后蛋白水平表达谱变化,并对蛋白活性进行了检测。Trx是一种广泛存在于生物体的具有氧化还原活性的酸性小分子蛋白质。松江鲈Trx1(TfTrx1) cDNA全长为665bp,包括5’端非编码区39bp,3’端非编码区290bp,开放阅读框336bp,编码111个氨基酸。TfTrx1预测分子量为12.34kD,理论等电点(pI)为4.52。TfTrx1不含信号肽,与裸盖鱼的同源性为66%,序列中含有高度保守的活性位点序列CGPC (Cys-Pro-Asp-Cys)。二级结构含有5个β折叠和4个α螺旋,TfTrx1蛋白结构与人类Trx1结构非常吻合。通过Neighbor-j oining方法构建的系统进化树显示,松江鲈Trx与裸盖鱼等鱼类Trx1首先聚在一起,然后与其他脊椎动物Trx1聚为区别于Trx2的一大支,从而也证明我们克隆到的是松江鲈Trx1基因。RT-PCR检测发现,TfTrx1在血、心、肝脏、鳃、胃、肠、皮肤、肌肉、肾、脾、脑、卵均有表达。LPS刺激后皮肤、肝脏、脾、和脑中TfTrx1转录水平存在明显的上调。在蛋白水平,皮肤和肝脏中,LPS刺激后TfTrx1的上调表达比mRNA水平的上调持续更长时间。重金属刺激组,在所有检测的组织(皮肤、肝脏、脾、鳃和脑)中,TfTrx1均在刺激后24h上调至最高峰。为进一步探讨其生物学功能,我们构建了TfTrx1重组质粒,获得了纯化重组蛋白。荧光活性检测发现,TfTrx1蛋白二硫键的还原能够将色氨酸的荧光强度提高近2.6倍,这暗示色氨酸残基周围微环境的变化。胰岛素二硫键还原酶活性检测发现,TfTrx1蛋白在体外具有还原酶的活性,且活性具有一定的浓度依赖性。这说明TfTrx1在生理条件下,可作为一种氧化还原酶,参与机体的免疫应答过程。LPS作用实验发现,TfTrx1可以与LPS直接结合发生相互作用。细菌结合和凝集实验发现,TfTrx1可能通过与LPS的作用而导致其具有结合和凝集革兰氏阴性菌的活性。这暗示TfTrx1可能在抵抗LPS对组织、器官损伤,促进吞噬细胞吞噬作用等机体免疫防御中发挥作用。TRP14是具有Trx样活性中心的硫氧还蛋白超家族成员。松江鲈TRP14(TfTRP14) cDNA序列全长945bp,开放阅读框为372bp,编码由123个氨基酸组成的蛋白质。蛋白的预测分子量为14.11kD,理论等电点(pI)为5.45,没有信号肽。多序列比对结果显示TRP14具有高度保守的活性位点序列CPDC (Cys-Pro-Asp-Cys), TfTRP14与裸盖鱼序列同源性高达90%。二级结构含有5个β折叠和4个α螺旋,TfTRP14蛋白结构与人类TRP14结构非常吻合。与TfTrx1相比,TfTRP14活性位点周围包括一个延伸的α2-β2环和一个α3a折叠区,这可能会导致两种蛋白底物的特异性。实时荧光定量结果显示,TfTRP14在各个组织均有表达,但是表达量高低具有组织特异性。LPS和重金属刺激后,TfTRP14表达存在明显上调,表明TfTRP14可能参与到松江鲈机体对微生物和重金属的免疫防御中。为了验证TfTRP14生物学活性,利用原核表达系统获得TfTRP14重组蛋白,活性二硫键的还原能够显著的提高色氨酸残基的荧光强度,但是胰岛素二硫键还原酶活性显著的低于TfTrx1,可能由于TfTRP14与TfTrx1具有不同的氧化还原特点,因而使底物具有一定的差异性所致。由于绝大部分过氧化物还原酶在体内以硫氧还蛋白作为惟一的氢供体,因此又被称为硫氧还蛋白过氧化物酶。松江鲈Prx1(TfPrx1)的cDNA序列全长1047bp,开放阅读框为600bp,编码199个氨基酸。TfPrx1预测分子量为22.35kD,理论等电点为6.42。多序列比对发现,TfPrx1含有Prx特征肽段"FYPLDFTFVCPTEI"和"GEVCPA",属于典型的2-Cys型Prx。系统发育树显示松江鲈Prx与其他物种的Prx1聚在一起,区别于Prx2分支,这证明我们克隆到的是松江鲈Prxl基因。TfPrx1在检测的各个组织中都有表达,其中以肠中表达最强,其次为脑,而在肝脏和肌肉中的表达较弱。LPS和重金属刺激后,TfPrx1在主要免疫器官和脑中均明显上调表达,表明TfPrx1可能在抗氧化应激相关的机体免疫中发挥重要作用。该基因的原核重组表达蛋白在DTT存在的条件下表现出较强的过氧化物酶活性,对超螺旋DNA有较强的保护作用。这些结果说明,TfPrx1作为一种重要的抗氧化酶,在松江鲈体内能够有效的抑制ROS引起的机体氧化损伤。更多还原

【Abstract】 Roughskin sculpin, Trachidermus fasciatus Heckel (Scorpaeniformes:Cottidae), is a carnivorous fish with seawater-freshwater migratory habits. As one of the second order of protected aquatic animals in China, roughskin sculpin is of great biological, ecological, and economic importance. Considering its serious decline during the last few decades, aquaculture has become an important strategy for recovering its population in the wild. However, roughskin sculpin possesses unique pathologic feature due to its unique biological and physiological characteristics. Up to now, few studies have reported on the molecular mechanisms of innate immunity in roughskin sculpin. It has been reported that reactive oxygen species (ROS) are highly produced in the process of microbial invasion or heavy metal exposure. ROS may be helpful to the host in the process of killing invading bacteria at low concentrations. On the other hand, the elevated levels of ROS could cause cytotoxicity to host cell and the immune system. Thus, to eliminate of these excessive ROS and maintain redox balance is of great importance for the proper function of cells and innate immune response of organisms.In the present study, thioredoxinl (designated TfTrxl), thioredoxin related protein14(designated TfTRP14) and peroxiredoxinl (designated TfPrxl) were cloned from roughskin sculpin using rapid amplification of cDNA ends (RACE) approaches. Quantitative real-time PCR (qRT-PCR) was employed to assess the mRNA expression of the three genes in various tissues and their temporal expression in important immune organs and brain of roughskin sculpin post E.coli LPS challenge and heavy metal exposure. To elucidate their biological functions, they were expressed in E. coli BL21(DE3).Trx is a small multifunctional protein ubiquitously expressed in bacteria, plant, and animals, which has a redox-active disulfide/dithiol within the conserved active site sequence CGPC (Cys-Pro-Asp-Cys). The full length cDNA of TfTrxl is665bp with a336bp ORF, encoding a protein of111amino acids with the predicted molecular weight of12.34kD and isoelectric point (pI) of4.52. No signal peptide was detected in the protein. The similarity was66%between roughskin sculpin and sablefish (Anoplopoma fimbria). The active sites CGPC were highly conserved according to the multiple sequence alignment. It has five β-sheets and four a-helixes, and the active sites locate on the surface of the protein. NJ tree reconstructed showed that TfTrx1clustered to Trxls from other fishes, in accordance to their traditional taxonomy. qRT-PCR analysis revealed the presence of TfTrx1transcripts in all detected tissues including blood, heart, liver, gill, stomach, intestine, skin, muscle, kidney, spleen, brain and ovary. The expression of TfTrx1mRNA in the skin, liver, spleen and brain was up-regulated post LPS challenge. In the skin and liver, the TfTrx1protein was significantly induced detected with western blot and maintained much longer time than the increase of TfTrx1mRNA. In the heavy metal exposure experiment, all the detected tissues (skin, liver, spleen, gill and brain) showed an increased expression. These results suggested that TfTrx1may be involved in the innate immunity related antioxidant response of roughskin sculpin. To investigate the biological activities of TfTrx1, a recombinant protein were constructed using an E. coli expression system. TfTrx1, which contains W34(Tryptophan, Trp or W), yielded a2.6-fold increase in tryptophan fluorencence when reduced, suggesting changes in the microenvironment around the W34residue. Furthermore, TfTrxl exhibited a concentration-dependent disulfide reductase activity towards insulin. These indicated that TfTrx1may play roles in fish immune response against oxidative damage in physiological state as a redox enzyme. In vitro incubation of TfTrx1with LPS demonstrated that TfTrx1could directly interact with E. coli LPS. Furthermore, TfTrx1could agglutination and binding gram-negative bacteria, possibly via the interaction with LPS. Thses suggested that TfTrx1may protect against LPS-induced tissue injury, and play role in the promoting phagacytosis of invaded pathogen.TRP14is a member of Trx superfamily with a Trx-like motif CPDC (Cys-Pro-Asp-Cys). The full length cDNA of TfTRP14is945bp with a372bp ORF. It was translated to a123amino acid protein with molecular weight of14.11kD and isoelectric point (pI) of5.45. The active sites CPDC were highly conserved from the multiple sequence alignment. The similarity was up to90%between roughskin sculpin and sablefish. It has five β-sheets and four α-helixes, the same as TfTrxl. However, the surface near the active sites of TfTRP14included an additional helix α3a and an extended β2-α2loop, which protruded, as characteristic of human TfTRP14. This may cause substrates difference between TRP14and Trx. TfTRP14was broadly expressed in all the examined tissues, with the highest transcript level in the brain and skin. The expression of TfTRP14was up-regulated post LPS challenge and heavy metal exposure, implying the involvement of TfTRP14in fish immune related antioxidant response. In addition, a fusion protein containing TfTRP14was produced in E. coli expression system. TfTRP14, which contained W42, W77yielded a5.4-fold increase in tryptophan fluorencence when reduced, higher than that of TfTrxl (2.6-fold), suggesting changes in the microenvironment around both W residues. In insulin reduction experiment, TfTRP14demonstrated a concentration-dependent disulfide reductase activity significantly lower than TfTrx1. This might be due to the substrates difference between these two proteins resulted from different redox characteristic.Prxs are abundant cellular antioxidative proteins which play important roles in protecting organisms against the toxicity of ROS. The full length cDNA of TfPrxl is1047bp with an ORF of600bp, encoding a protein of199amino acids. The predicted molecule weight is22.35kD and isoelectric point (pI) is6.42. Multiple sequence alignments demonstrated the presence of two characteristic peptides of Prx "FYPLDFTFVCPTEI" and "GEVCPA", indicated that TfPrxl was a classic2-Cys Prx. NJ tree showed that TfPrxl clustered with Prxls from other species, apart from Prx2s, indicating that rouskin sculpin Prx belonged to Prxl. TfPrx1ubiquitously expressed in all the detected tissues, with the most abundant expression in the intestine, followed by the brain. In the brain and main immune organs, the expression of TfPrxl was significantly induced post LPS chanllege and heavy metal exposure, suggesting that it play an important role in the innate immunity and antioxidant activity of roughskin sculpin. The recombinant protein of TfPrxl demonstrated the capability to reduce the H2O2when dithiothreitol (DTT) was used as a reducing equivalent and to protect DNA from oxidative damage. These findings suggested that TfPrx1probably play an essential role in antioxidant defense in physiological state.更多还原